Biol 567L: Biochem, Cell & Mol Bio Lab II

Chem 567L - Biochemistry, Cell & Molecular Biology Laboratory II
Fall 2010
Instructor: Anca Segall
Office/lab: LS 331, 619-997-4490
email: asegall@sunstroke.sdsu.edu
Co-Instructor: Jason Rostron
Lab: LS 331, 619-594-6528
email: 1220jer@gmail.com
LS420
4:00-6:40 pm (optimistically – you will spend a lot more time in this lab)
Class Objectives
This course is designed to teach techniques commonly encountered in graduate school and
biotech. The class is divided into three sections, and 3 research projects. In the first segment, you
will learn about genomic sequencing approaches and using multiplex PCR to “close” genomes.
In the second segment of the class, you will perform a research project combining genetics,
molecular biology, cell physiology, and biochemistry. In the third section you will
design and conduct your own experiments. Unlike other laboratory courses, you will be
responsible for making solutions, troubleshooting protocols, and interpreting the results. You
will work in groups of 2 - 4, but you are expected to develop your own skills.
The overall goal of the course is to train you to become independent in your thinking and in the
application of your skills.
Section 1 - Characterization of microbial communities for Bacteria by 16S rDNA cloning
and sequencing
1 - Isolate genomic DNA from bactéria
2 - PCR 16S rDNA as a positive control of primer sets of 12 – 24 primers to identify segments
that bridge gaps in a bacterial genome sequence
3 – Purify segments and clone with TOPO-TA kit
4 - Sequence clones
5 – Perform alignment of sequences with rest of genome
Brief Lectures
The 16S rDNA loci and uncultured microbes
Principles of DNA isolation
Principles of primer design – multiple strategies for different uses
Newest methods of DNA sequencing
Cloning - plasmids, cosmids, BACs, enzymes
PCR and sequencing (potential trip to SDSU sequencing facility)
Bioinformatics - BLAST, NCBI, ribosomal database, the SEED – Ramy Aziz or Anca Segall
Other useful databases and sites
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Practicals
Calibration of pipettors
Solution preparations
Autoclave and plate pouring
PCR
Polyacrylamide gel electrophoresis
TOPO and SLIC cloning
Bioinformatic analyses of 16S rDNAs
Section 3 - Design and conduct independent research.
You will discuss your ideas for this segment with Anca Segall or Jason Rostron, and get help
with designing an experiment that will give meaningful results. However, you will be expected
to think about and come up with ideas on your own first.
Required Materials:
Lab notebook – a normal compositions book will be fine
Lab coat
Fine-tip Sharpie
Calculator
Lab notebooks:
Laboratory notebooks will be periodically checked as part of your subjective grade (20 points
total). These notebooks can also be used during the practical exams, so it is to your
advantage to do a good job.
Short write-ups and pop quizzes:
Pop quizzes and short write ups will be given throughout the course of the semester on material
already covered and also on new material that you should have read before coming to
class (up to 100 points).
Practical Exams:
You will obtain up to 30 points for the practicals
Write-up of Second section project
This write-up will serve as the practice for the final project report. It is worth 30 pts.
Presentation and write-up of Final Project:
10-12 minute powerpoint presentation AND a report of your independent project (100 points).
Presentations can be done as a group; reports are independent efforts.
Attendance:
You must attend each class meeting. If you miss a class, let me know why and when. If you miss
2 classes you will be dropped or you will fail.
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Grading Scale:
94 - 100% A 74 - 76% C+ 60 - 63% D
90 - 93.9% A- 70 - 73% C below 60% F
84 - 89.9% B+ 67 - 69% C-
80 - 83% B 64 - 66% D+
77 - 79% B- 60 - 63% D
Laboratory Notebooks:
Notebook Table of Contents: Experiment title, date, and page numbers
Title: State the title of the experiment.
Purpose: State the purpose of the experiment. Why are we doing the experiment?
Data/Calculations/Results: Include all data: figures, tables, calculations, etc. Introduce each
result section.
Discussion/Conclusions/: State any conclusions you have regarding data and why or why not
expected results were obtained. Example: As expected, relatively pure, undegraded RNA was
isolated from mammalian cells as seen in Figure 1, Lanes 1-3, which represent 1, 5 and 10µl of
mammalian RNA, respectively.
Future: I will change X in order to get better results; or, I will do Y to answer the question that
arises from my last experiment.
Tape protocols, disks with data, Post-It notes into your notebook. The main purpose of the
notebook is to be understandable at a latter date....
DO NOT ERASE OR WHITE-OUT MISTAKES; cross out with a line or an X
DO NOT COVER OVER ANY TEXT
Schedule for Fall 2010 (Monday and Wedesdays)
Week 1 - Check in and Starting Quiz
- Autoclave, pour plates with antibiotics
- Genomic DNA isolation
- Pipettors, calibrations, making solutions, and dilution problems
Week 2 – 5
Week 5 – 9
(note overlap)
Week 8 – 9
- First project: closing a genome using multiplex PCR
- Second project, starting with SLiC cloning
- Students will plan and discuss independent projects with instructors
Week 10 – end - Independent (third) project
Last day in lab and check-out: December 8;
Dates for presentations to be determined; all independent 3 rd project reports due December 16
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